STUDIES ON THE MORPHOLOGY AND MOLECULAR CHARACTERIZATION OF STORED GRAIN WEEVILS SITOPHILUS (CURCULIONIDAE: COLEOPTERA)

In this paper , Sitophilus oryzae, Sitophilus zeamais , and Sitophilus granarius , three economically significant grain pests are thoroughly analyzed in terms of their morphological and molecular differences. The work provides critical insights into species identification, genetic differentiation, and adaptation by contrasting conventional morphological features and genetic approaches. The samples were collected from eight talukas of district Khairpur: Kingri, Kot Deji, Khairpur, Gamabat, Mirwah, Sobhodero, Nara, and Faizganj during the year 2018-2022. The samples were identified on the basis of morphology and extraction of DNA was done. The samples were sent for sequencing. The findings provide important evolutionary insights and aid in pest management. Additionally, the text describes the distinguishing physical characteristics of mature Sitophilus granarius , maize weevils, and rice weevils, emphasising size, colour, markings, and anatomical aspects. Understanding the differences among various weevil species is made easier by this information.


Introduction
The Sitophilus genus includes several significant stored grain pests, such as the rice weevil and the maize weevil, Sitophilus oryzae and Sitophilus zeamais.Due to their propensity for destruction and capacity to invade and harm stored grains, specifically rice and maize.These two species are of significant economic importance.However, due to overlapping characteristics in their identification, it is essential to distinguish between these two weevil species in order to design successful pest management plans and focused control actions (Hagstrum and Subramanyam, 2019).Some of the prevalent and harmful pests of stored cereals belong to the genus Sitophilus Schoenherr.The tropics, subtropics, and warm zones are home to large populations of the rice weevil Sitophilus oryzae (L.) and the maize weevil Sitophilus zeamais (Motsch.).Both may grow on a variety of grains, although Sitophilus oryzae prefers wheat while Sitophilus zeamais prefers maize (Bernal and Navarro, 2013).However, new research by Athanassiou et al. (2017) shows that although popular names of these species are connected to certain grains, it does not necessarily imply that these grains are the only the best candidates for their development.Additionally, it is said that adults of S. oryzae and S. zeamais were much more numerous on rice than on maize.The FAO estimates that 10% of the world's grain is lost each year.Whole grain infestations can result in weight loss, fungal development, and quality loss due to an increase in free fatty acids (Trematerra, 2009).The invasion of these species could make it easier for mites, infections in stored products, and secondary colonizers to establish themselves.Effective control of these pests requires knowledge of their biology (Smith and Smith 1989).Few writers who have conducted biological research on specific Sitophilus species have noted variations in the fertility of S. oryzae on host plants such rice, wheat, sorghum, and maize.Additionally, it was shown that animals raised on wheat had shorter developmental times and longer life spans (Folmer et al., 1994;Smith and Smith 1989;Smith and Navarro, 2017).This study also revealed specifics regarding S. oryzae's developmental cycle's seasonal variations.According to Makarova and Kuzmina (2016), who conducted a comparative study, there is a significant variation in body size between the two Sitophilus species in terms of their distribution, body size, food preferences, hibernating habits, field infestation, heating, developmental period, and flying ability.Despite their significant prominence as stored grain pests, Smith and Smith (1989) cites biological research of the Sitophillus species and claims that no current or thorough analysis whereby their physiology is contrasted has been presented.According to Shi et al. (2007), S. oryzae and S. zeamais have different developing periods.It was found that for S. oryzae, oviposition and the developmental period were more favourable in wheat than for S. zeamais.The biological characteristics of each of the three Sitophilus species were presented by Mason and McDonough in (2012).The variation and differentiation between S. oryzae and S. zeamais have been extensively studied using morphological and molecular approaches.According to Cruickshank (2002), molecular markers utilized in phylogenetic investigations should have certain characteristics that make them appropriate for resolving the specific taxonomy level or ranking under examination.The requirement for an amino acid substitution or mutation frequency that impacts an appropriate balance among producing a sufficient number of useful (variables across sequences) sites and avoiding long saturating of the nucleotide locations, that could mask genetic communication and is one of the most essential of these (Mason and McDonough, 2012).To ensure that sufficient relevant changes are present during the relatively brief period that these genera have been diverging, studies looking at very closely related organisms will need genes with quick substitution rates (for example, at the species-level within a genus).In contrast, genes with slower substitution rates are needed when examining organisms that are more distantly related (such as at the family-level or above), as this will reduce the effects of saturation and provide evidence for these older divergences (Cognato and Sperling, 2000).Paralogous sequences are less likely to arise when a gene is present as a single copy or several homogenous forms on haploid chromosomes, rather than when different copies of the gene are present in distinct individuals.While each cell contains several copies of the mitochondrial genes.Originally, this gene has been used (though not exclusively) in smaller-scale phylogeny to investigate associations between newly diverging, rather closely related species due to its high substitution rate (Cognato and Sperling, 2000).Weevil classifications at the level of genera have used Cox1 sequences, either on their own or in combination with other genes (as in the scolytine genus Ips phylogeny), for example, an additional MtDNA gene and two nuclei genes.There may be less evolutionary help for internal nodes compared to peripheral clades if the studied group is too divergent and cox1 alone is ineffectively too saturated to resolve internal nodes.
Despite its limited utility in resolving phylogenies of distantly related species, Cox1 has been shown to be useful in other contexts.In this paper, we examine and discuss the research that has been done about the morphological and molecular differences between Sitophilus oryzae, Sitophilus zeamais, and Sitophilus granarius.Present study provides a thorough summary of the most recent research on the taxonomic, genetic, and evolutionary features of these three economically significant weevil species by synthesizing the results from numerous investigations.

Materials and methods
Sitophilus species were collected for the current study from different of locations in Khairpur, Sindh, Pakistan.With the aid of farmers, the weevils were removed manually from palms that were affected.For this investigation, 210 adult weevils' total 105 males and 105 females, were collected.Sample collection: Sitophilus species were collected from Khairpur, Sindh, Pakistan.The weevils were captured from infested palm trees using handpicking methods.The samples were collected from eight talukas of district Khairpur: Kingri, Kot Deji, Khairpur, Gamabat, Mirwah, Sobhodero, Nara, and Faizganj.Morphometric measurements: A morphometric analysis of the weevils is performed by utilising a pair of calipers and a binocular stereo microscope to study their various parts of body i-e: abdomen length (al), Body length (l), pronotum width (pw) pronotum length (pl), head size (hs), the distance of the tip of the rostrum to insertion of the antennae (ta) , and abdomen width (aw) are all included (Mizzi et al., 2009Panhwar et al., 2023) Molecular study DNA Extraction: The COI-1 (Cytochrome C Oxidase) gene is frequently used as a DNA barcode to distinguish between various species.Due to the conserved sequence of COI in conspecifics, it is employed to extract since it is found in mitochondria.The QIAamp DNA Minikit was used to help with the DNA extraction process.For the full extraction of mitochondrial and genomic DNA from tissues, utilize the QIAamp DNA Minikit (Black et al. 2001;Brown et al.1997;Armstrong and Wratten, 1996).

Polymerase
Chain Reaction (PCR): PCR amplification of the COI gene region was done.Created and picked suitable primers that amplify the COI gene's conserved sections in order to guarantee effective amplification in many species.For COI amplification in insects, commonly used primer pairs included HCO2198 and LCO1490.PCR Amplification Protocol: Using the extracted DNA as a template, set up PCR reactions in a sterile setting.The following general cycling conditions were utilized for PCR (particular parameters may differ depending on the thermocycler and primers used): Initial denaturation: 3-5 minutes at 95°C Cycling (25-35 cycles): Denaturation: 30 seconds at 95°C Annealing: 30 seconds at the appropriate primerspecific temperature (typically around 50-60°C) Extension: 1-2 minutes at 72°C Final extension: 5-10 minutes at 72°C DNA Sequencing: Send the PCR products for Sanger sequencing after purifying them.The COI gene's actual DNA sequences for each specimen of the weevil were be generated in this step.

Phylogenetic and Blast Analysis
Prior to sequencing, PCR-amplified products underwent purification.In order to sequence the cytochrome oxidase subunit-1 and internal transcribed spacer regions, forward and reverse primers were used.Weevils obtained from several locations within the Khairpur district were used to choose these areas.The Khairpur weevil sample-derived PCR products were effectively used to produce sequences.Sequences with useless or empty information were manually removed until the base pairs were aligned in their final position.
Type species: Curculio oryzae (Linnaeus, 1763): Diagnosis.One of the notable pests of stored products, grains, and seeds are the fauna that make up the genus Sitophilus.Sitophilus oryzae, a significant pest of cereal grains and goods, is one of the most globally distributed species of rice weevil, and its larvae develop inside a seed kernel or similar items.In the genus Curculio, Sitophilus oryzae was initially proposed by Linnaeus.Subsequently, other synonymies were proposed.Last but not least, these stored grain pests were classified as Sitophilus by Schoenherr 1838.Due to its striking physical resemblances to the rice weevil, this species has historically been mistaken for another or thought to be a morph.

Morphological description of Sitophilus oryzae (Linnaeus, 1763):
The pronotum, the shield behind the skull, is punctured with coarse, circular holes, and the head has a thin snout.The elytra have sharp punctures and deep lines.The snout is nearly one millimetre long.The prothorax or elytra are as long as the head including the snout.The elytra feature lines of holes inside transverse grooves, whereas the prothorax (the part of the body above the scalp) is heavily pitted.Weevil adults measure between 3.1 and 4.3 mm.The adult rice insect is a dark reddish-brown to black in color, with 4 faint red or yellow spots on the elytra (wing covers) and round or irregularly shaped pits on the thorax (Fig. 1).

Worldwide Distribution of Sitophilus oryzae:
Sitophilus oryzae is distributed in India, Brazil, Srilanka, Pakistan, New Zealand, Australia, China, Cuba, United Kingdom, Sweden, Spain, Canada, Poland, Germany, Russia, Italy, Africa, Egypt, Greece, Kenya, Malaysia, Israel and Iraq.Sitophilus oryzae distribution in Khairpur.:This species is found extensively throughout the different talukas of the Khairpur district.The taluka Gambat had the highest population of this species, then following Nara as well as Khairpur.The taluka Faiz Ganj has the lowest number of Sitophilus oryzae, followed by Kot deji as well as Mirwah.Besides, taluka Kingri as well as Sobho dero also had a medium population of Sitophilus oryzae (Fig. 2 & Table.1).

Sitophilus granarius distribution in Khairpur:
Sitophilus granarius is found extensively throughout the different talukas of the Khairpur district.The taluka Nara had highest population of this species, then following Kot deji then Kingri.The taluka Khairpur had the lowest concentration of Sitophilus granarius, which proceeded by Mirwah as well as Faizganj.Additionally, taluka Gambat and Sobho dero both had a medium population of Sitophilus granarius (Fig. 5 & Table .4). the Gene Bank were used for alignment by BioEdit software, and a phylogenetic tree was created in MEGA6 to show the relationship of Sitophilus granarius with previously reported species in the NCBI (National Centre for Biotechnology Information).The results of the current study revealed strong genetic similarities to the Sitophilus granarius species found in the USA, accession number (MG458963.1)depicted in Figure 6.

Morphological description of Sitophilus zeamais
Motschulsky 1855: On the wing coverings, the reddish patterns are more distinct.The maize weevil is a small, variable-sized snout beetle with an average length of four millimetres.It can range in colour from dark reddish brown to practically black, and the back normally bears four faint reddish or yellowish patches.Under its wing covers, the maize weevil has fully grown wings and is able to fly with ease.With the exception of a smooth, narrow strip running down the centre of the dorsal (top) side, the thorax is heavily pitted and punctured with slightly irregularly shaped holes (Fig. 7).

Worldwide Distribution of Sitophilus zeamais.
This species occurs in Australia, Korea, Africa, India, Russia, Egypt, Japan, Yemen, Italy, Syria, Saudi Arabia, Sri lanka, Pakistan, Malaysia, China, Germany, Brazil, Iraq, New Zealand, Greece, Canada, Poland Cuba, Sweden and United Kingdom.

Sitophilus zeamais distribution in Khairpur.
Sitophilus zeamais is distributed throughout the different talukas of the Khairpur district.The taluka Nara had the highest population of this species, then Kingri as well as Sobhodero.The taluka Khairpur had the lowest number of Sitophilus zeamais, which was subsequently pursued by Mirwah then Gambat.Besides, taluka Kot deji and Faizganj both have a medium population of Sitophilus zeamais (Fig. 8 & Table .7)Phylogeny of Sitophilus zeamais: The cytochrome oxidase I (COI) gene's nucleotide sequence was taken from Sitophilus zeamais and analysed using BLAST analysis.Following that, pertinent sequences obtained from the Gene Bank had been utilised for alignment by BioEdit programmes, and a tree of phylogeny was generated in MEGA6 to show the relationship of Sitophilus zeamais with previously identified species in NCBI.The current analysis revealed strong similarities to the Sitophilus zeamais species described from China and assigned the accession number (MT294139.1) in Figure9  Weevils are members of the superfamily Curculionidea, which also includes several widely recognized Coleoptera species.Several weevils possess snouts that are the same length as their bodies (Farrell et al., 2001).Most weevil species that prey on grains belongs to genus Sitophilus.Weevil infestations are periodic or temporal and fluctuates between numerous months of the year, according to various survey programmes (Wakil et al.,2021).During the present study Sitophilus species were collected from eight talukas of district Khairpur: Kingri, Kot Deji, Khairpur, Gamabat, Mirwah, Sobhodero, Nara, and Faizganj.A total of 210 samples of weevils were captured from infested palm trees using by handpicking methods.The samples were identified into 03 species: Sitophilus oryzae, Sitophilus zeamais, and Sitophilus granarius.Zafar et al., (2021) while studying the diversity of weevils in Gujrat, Pakistan collected 315 specimens of weevils and reported 03, i-e: Sitophilus granarius, Sitophilus zeamais.They also noted that diversity was consistent throughout, temperature and humidity had an impact on abundance.The abundance rose along with these two characteristics.August having the highest abundance.More female weevils than male weevils were present.Between the two factors and diversity, a positive link developed.Most of weevils, at larvae and adults stage, feed on plants.Weevils are significant agricultural pests because they chew holes in fruits, nuts, and other plant material.The majority of the adult weevils possess a long rostrum and use to help ovipositing the eggs (deposit eggs) in plant tissue.Others feed on decaying wood or bark, while certain wood-borers eat ambrosia fungus (Farrell et al., 2001).Infestations of stored grain insects can lead to decreased weight, quality, earnings, and seed viability.The majority of these insects about 75% are coleopterans, and the most destructive species of storage insects are found in the genera Sitophilus and Tribolium.The economically important and widespread bug Sitophilus species consumes stored grains.The outermost layer of the grain is not the preferred food source for the larvae, which causes a large loss of protein as well as vitamins.The adults of weevils mostly consume its endosperm lowering the amount of carbohydrates present.Insects which attack the germ particularly will suffer a greater loss in germination (DalBello et al., 2000).Besides, morphological features.Cox1 has emerged as the preferred gene for DNA barcoding initiatives.It's critical to differentiate among phylogeny, which aims at reconstructed history of evolution, and barcoding, and whose goal is to identify species.Cox1 is especially well suited for this because it is present in all eukaryotes, has a high mutation rate, and lacks introns.Additionally, there are universal primers that make it relatively simple to amplify the gene across many lineages.It has been demonstrated to be reliable and economical.Cox1 sequences have recently been employed with effectiveness for automatic species finding and delimitation (Hebert et al., 2003).During the present study the Cytochrome Oxidase I (COI) gene's nucleotide sequence was taken from the Sitophilus oryzae, Sitophilus granarius, and Sitophilus zeamais were subjected to BLAST analysis.Then, relevant sequences from the Gene Bank were used for alignment by BioEdit computer programmes, and a tree of phylogeny was built in MEGA6 to show the relationship of the Sitophilus oryzae with previously reported species in NCBI.The current analysis revealed strong similarities to the Sitophilus oryzae species, which was discovered in India and assigned the accession number (KF298426.1).Sitophilus granarius provided the nucleotide sequence for the Cytochrome Oxidase I (COI) gene, which was then subjected to BLAST analysis.Relevant sequences from the Gene Bank were used for alignment by BioEdit software, and a phylogenetic tree was created in MEGA6 to show the relationship of Sitophilus granarius with previously reported species in the NCBI (National Centre for Biotechnology Information).The results of the current study revealed strong genetic similarities to the Sitophilus granarius species found in the USA, accession number (MG458963.1).The cytochrome oxidase I (COI) gene's nucleotide sequence was taken from Sitophilus zeamais and analysed using BLAST analysis.Following that, pertinent sequences obtained from the Gene Bank had been utilised for alignment by BioEdit programmes, and a tree of phylogeny was generated in MEGA6 to show the relationship of Sitophilus zeamais with previously identified species in NCBI.The current analysis revealed strong similarities to the Sitophilus zeamais species described from China and assigned the accession number (MT294139.1).This study has confirmed the status of Sitophilus species both on morphology and molecular basis.

Conclusion
Present study concluded that based on their morphology and DNA makeup, the current study has determined that the three species of the genus Sitophilus that are commonly referred to as stored grain pests are actually three distinct species.Additionally, this is the only piece of its kind to come from Pakistan's Sindh to the best of our knowledge.Hopefully, the results of this study will act as a starting point for future researchers that are interested in the Sitophilus fuana.

Fig. 3
Fig. 3 Phylogeny of rice weevils (Sitophilus oryzae) Morphological description of Sitophilus granarius (Linnaeus, 1875): Sitophilus granarius adults can range in length from 2.9 to 3.9 mm, while 3 to 4 mm is most common.They display the Curculionidae family's distinctive rostrum and elbowed antennae.They have an evenly polished tint ranging from reddish brown to lustrous black.Short, robust yellow hairs are sporadically distributed throughout the body.The head extends into a thin snout.In comparison to females, men have closer and more pronounced punctures on the dorsal surface of the rostrum.There are prominent round punctures on the prothorax.Adults are unable to fly because they lack wings (Fig 4).

Table 3 Morphometry of Sitophilus oryzae (Female)
Phylogeny of Sitophilus oryzae:The Cytochrome Oxidase I (COI) gene's nucleotide sequence was taken from the Sitophilus oryzae and subjected to BLAST analysis.Then, relevant sequences from the Gene Bank were used for alignment by BioEdit computer programmes, and a tree of phylogeny was built in MEGA6 to show the relationship of the Sitophilus oryzae with previously reported species in NCBI.The current analysis revealed strong similarities to the Sitophilus oryzae species, which was discovered in India and assigned the accession number (KF298426.1) in Fig 3.

Table 6 Morphometry of Sitophilus granarius (Female).
Phylogeny of Sitophilus granaries:Sitophilus granarius provided the nucleotide sequence for the Cytochrome Oxidase I (COI) gene, which was then subjected to BLAST analysis.Relevant sequences from